Macrophages play a key role in wound repair and fibroproliferation by producing cytokines and growth factors that regulate both inflammation and angiogenesis. Macrophages are exquisitely sensitive to their micro-environment, and production of cytokines and growth factors is tightly regulated. We have discovered a novel signaling pathway in macrophages that results in the strong up-regulation of VEGF and downregulation of TNFalpha and IL-12 expression, constituting an angiogenic switch. This pathway involves a synergistic interaction between Toll-like receptor (TLR)-2, 4 and 9 agonists and adenosine A2A receptor (A2AR) agonists. This grant will study the basis for the synergistic interaction between these disparate signaling pathways. Aim 1: Co-immunoprecipitation techniques will be used to determine whether physical interaction occurs between A2ARs and TLRs. Primary macrophages and RAW264.7 cells transfected with epitope-tagged expression constructs will be used. Aim 2: The role of down-stream signaling components of the TLR pathway (MyD88, IRAK-1, IRAK-4, IRAK-M) will be studied, using knockout mice, and transfection of dominant negative transcripts into RAW264.7A2A cells. Aim 3: Down-stream signaling components of the A2AR pathway will be studied. The role of G-protein sub-units (Gsalpha and Gi), and adenylyl cyclase activation and cAMP production in the synergistic interaction will be studied. Aim 4 will determine whether the expression of genes other than VEGF is regulated by the interaction between A2ARs and TLRs. Initial studies using Affymetrix gene chips indicate that this pathway regulates a small group of genes. We will extend and confirm the gene chip analysis, and then perform proteomic analysis using 2-D Difference Gel Electrophoresis and MALDI-TOF MS, to analyze proteins that are differentially regulated by this synergistic pathway. Aim 5 will examine the mechanism of down-regulation of TNFalpha by A2AR agonists in TLR-agonist-treated macrophages. The level of regulation (transcriptional, post-trancriptional, mRNA stability), and the role of NF-KappaB activation will be studied. Aim 6 will study the role of the synergistic interaction between A2ARS and TLRs in regulating wound healing and angiogenesis in vivo. The effects of A2AR agonists and antagonists on excisional wounds in MyD88 mice (deficient in TLR signaling) will be analyzed. These experiments should help to clarify the significance of this synergistic interaction in vivo.